Stem cell research is studying the principles of tissue regeneration processes in order to develop methods for regenerative medicine. One very important factor of stem cell biology is the constant communication between the stem cells themselves and the interplay of the stem cells and the surrounding tissue, the so called stem cell “niche”. Together, these cells form organizational units, cell clusters or “microorgans” that in large number and sophisticated architecture ultimately form an entire organ.
These processes are being studied in various experimental settings of which one of the most classical ones is the use of “hanging drops,” where stem cell development can be simulated by putting a certain amount of stem (and other) cells together in a drop in a way that cell clusters develop which can be analyzed. One major disadvantage of this widely used technology is the limited number of cell clusters that can be generated and the impossibility of performing a medium change, which would be most desirable because stem cell differentiation is dependent on the sequential change in cytokine signaling which could be triggered by providing these cytokines with a medium change.
In clinical settings, the prospect of large scale production of cell clusters of defined size with the possibility of performing a medium change would be very desirable for various therapeutic approaches, such as islet cell transplantation. In this technique, small islets perform better than large ones because of the limited diffusion based nutrient and oxygen supply in the early post transplant period (Lehmann R. et al, Diabetes. 2007 March; 56(3): 594 603). It therefore would be desirable to make the large islets small. However, for successful production of small islets and clinical applications, islets would need to be dissociated into single cells and reaggregated to small “pseudoislets.” About 1,000,000 pseudoislets would be needed for transplantation, a number impossible to reach with hanging drop technology.
In United States patent application publications 2011/0086375 and 2010/0068793 a device for the production of cell aggregates is described. The device sold as Aggrewell (Stemcell Technologies, Vancouver, BC, Canada V5Z 1B3) is, however, of limited use for stem cell cluster production as well as for islet cell transplantation, because in stem cell cluster production the design of the ground cavities plays a major role due to the possibility of exogenally induced morphogen release and subsequent uncontrolled differentiation. In the device sold as Aggrewell, the cells are being pushed into pyramidal alignment due to the pyramidal cavity design with sharp tips, which can lead to the aforementioned morphogen release. Additionally, in this device the borders between the cavities have a width that allows single cells to rest on the borders, a state which needs to be avoided, again due to the possibility of uncontrolled cytokine release. Furthermore, this device does not have any defined medium change construction, which for stem cell applications would be very desirable as the sequential, rigorously defined application of various cytokines applied by medium changes is crucial for correct stem cell differentiation.
In islet transplantation, this device cannot be used because of the limited number of ground cavities per plate well; a plate with several thousand ground cavities per well would be needed in the art in order to make clinical applications possible. Additionally, in islet transplantation, the formation of clusters needs to be well supported by defined cavities because the microarchitecture of reaggregated pseudoislets in hanging drops resembles original islet architecture with similar spatial distribution of alpha, delta and beta cells which apparently have biological reasons (Cavallari, Moritz et al., ADA 2007 presentation number 2062 P). In the Aggrewell ground cavities, again, the sharp bottoms would push the cells into a non natural form with unknown biological and clinical consequences.
Besides Aggrewell, other groups also performed experiments on micron scale cavities and the cultivation of stem cells and generation of cell clusters (Khademhosseini 2006, Mohr 2006), but due to vertical sidewalls, widely spaced cavities and broad borders the cell cluster formation is not taking place in a controlled manner and results in substantial non uniformities. Future possible stem cell applications target the regeneration and/or replacement of damaged tissue. It is of utmost importance to assure that the differentiation of stem cells is rigorously controlled in order to avoid uncontrolled differentiation and hence tumor formation. Therefore, uncontrolled cytokine and/or morphogen release due to experimental conditions like the ones in the aforementioned devices need to be strictly avoided.
There is therefore a need in the art to provide a device that allows for the aggregation of cells, especially of stem and islet cells and preferably allows for the generation of uniform cell clusters with minimal differentiation or cell cluster formation disturbance by the cavity design. Additionally, the number of cavities should be high in order to produce substantial numbers of cell aggregates. Furthermore, the borders between the cavities should be as small as possible in order to avoid uncontrolled resting of single cells besides the clusters. Moreover, the device should be designed in a way that a controlled medium change is possible. All of these requirements are fulfilled by the present invention.